Many proteins such as lysozyme are formed from folded polypeptides which are held in place by disulfide bonds that link the folds. Analysis of proteins often involve restriction or cutting apart of the polypeptides by enzymes, such as trypsin, at specific locations of the protein. After the protein has been cut apart, specific sites on the protein can be studied. Sometimes, the specific locations where the enzyme is to cut apart the protein is inaccessible to the enzyme because of the folding.
One method for unfolding the polypeptides is to reduce the disulfide bond linking the folded polypeptides. Reduction is accomplished by adding a reducing agent to the protein. After the proteins have been reduced, typically they are alkylated to avoid reforming of the disulfide bonds. An alkylating agent is added to the protein to alkylate it. The protein is then prepared for restriction or cutting apart of the polypeptides by enzymes. Usually the reduction step involves incubating the protein sample at a specified temperature followed by multiple washings or desalting steps. After the washings, the alkylation step is carried out and also usually involves incubation followed by multiple washings or desalting.
Common prior art reducing agents used to reduce proteins include dithiothrediol (DTT) and tricarboxyethylphosphene, neither of which are volatile under a partial vacuum at around room temperature. One method of reducing a protein involves incubating the protein with one of a reducing agent followed by three washing steps. After the reducing step, the protein is then incubated with an alkylating agent such as iodoacetamide or iodoacetic acid, after which the sample is then again subjected to multiple washing steps. Lodoacetamide and iodoacetic acid are also not volatile under a partial vacuum at about room temperature. Although the method for reducing and alkylating proteins described above is common, it is tedious, time consuming, and provides multiple opportunities to lose all or a portion of the protein sample, especially when small samples are involved. The present invention is directed to decrease the amount of time involved in the reduction and alkylation of protein samples and reduce the risk of losing all or a portion of the protein sample.
One embodiment of the present invention includes a composition configured to modify proteins, the composition comprising a single volatile reagent. Another embodiment of the present invention includes a method of modifying proteins comprising the steps of providing a volatile reagent configured to reduce and alkylate the proteins, the reagent including a sufficient amount of solvent to substantially dissolve the proteins, and dissolving the proteins in the volatile reagent. Another embodiment of the present invention includes a kit for one-step reduction and alkylation of a protein sample, the kit including a volatile reducing agent configured to reduce the protein sample, a volatile alkylating agent configured to alkylate the protein sample, and a volatile solvent configured to substantially dissolve the protein sample.